This invention relates to a novel process of making carboxylated dextran and to improved carboxylated dextran made by the process of the present invention.
Iron dextran complexes have been widely used as injectable iron preparations for the prevention or treatment of iron deficiency anemias in animals and humans.
Dextran is a glucose polymer in which glucopyranose units are linked predominantly by alpha-1,6 linkage and to a lesser extent by alpha-1,2, alpha-1,3 and alpha-1,4 linkages. Crude, high molecular weight dextran is commercially obtained by growing Leuconostoc mesenteroides on sucrose substrate by methods known in the art. Dextran thus obtained is hydrolyzed and fractionated to yield low molecular weight dextrans.
It is known that effective complexation with iron is facilitated by introduction of carboxyl groups into the dextran molecule. Methods of preparing iron dextran complexes using carboxylated dextran are known.
For example, U.S. Pat. No. 3,536,696 to Alsop and Bremner describes a process for the preparation of a ferric hydroxide complex by reacting a suspension of ferric hydroxide with dextran heptonic acid formed by introducing a carboxylic acid group into the terminal unit of the dextran polymer molecule.
U.S. Pat. No. 4,370,476 to Usher et al describes the preparation of iron dextran complex by reacting a polycarboxylic acid dextran with ferric hydroxide to obtain a complex with comparatively high iron content. The polycarboxylic acid dextran is formed by first introducing into the dextran molecule, by a specific mild oxidation, a plurality of aldehyde groups which are subsequently converted to carboxylic acid groups by i) further oxidation of the aldehydes using stronger oxidizing agents or ii) cyanidation to produce the cyanohydrin derivative, followed by hydrolysis to produce the corresponding carboxylic acid.
U.S. Pat. No. 4,788,281 discloses that low molecular weight dextran (2000 to 6000) can be oxidized with sodium chlorite to produce a dextran hexonic acid derivative which will form a complex with ferric hydroxide having an iron content of up to 20% or more.
Although high iron content is possible in iron dextran complexes prepared using carboxylated dextran, one disadvantage is that patients receiving such preparations must be carefully monitored for shock and other clinical side effects. Moreover, in the case of iron complexes prepared using polycarboxylated dextran, the complexes are not completely stable at high temperatures such that autoclaving cannot be used to achieve terminal sterility.
It has now been discovered that the physicochemical stability and safety of iron dextran preparations made with carboxylated dextran can be improved if carboxylated dextran is reduced to remove all or substantially all reducing groups from the dextran prior to its complexing with iron.
In one aspect, this invention therefore relates to a novel process of preparing a carboxylic acid derivative of dextran comprising reducing a carboxylated dextran containing at least one carboxylic acid to remove all or substantially all reducing groups. The invention also relates to novel carboxylated dextran of the present invention which is non-reducing or substantially non-reducing and to metal complexes formed with the novel carboxylated dextran, including iron complexes.
In one embodiment, carboxylated dextran is reduced by hydrogenation to convert all or substantially all reducing groups to alcohol groups while leaving the carboxylated groups unaffected for complexing. Suitable methods of hydrogenating dextran by catalytic, electrolytic or chemical hydrogenation are described in U.S. Pat. No. 2,807,610 to Zief. Carboxylated dextran may be hydrogenated under similar conditions.
Most preferably, hydrogenation is effected by reacting carboxylated dextran with about 1 to 2% sodium borohydride in basic aqueous solution (pH 7.5 to 10.0), atmospheric pressure and ambient temperature. The concentration of sodium borohydride should be kept as low as possible to facilitate removal of residual borate ions after hydrogenation. Preferably, the minimum concentration of sodium borohydride sufficient to obtain carboxylated dextran which is non-reducing or substantially non-reducing is used.
Hydrogenation may also be effected by catalytic hydrogenation at 25xc2x0 to 60xc2x0 C. and 1 to 5 atmospheres pressure using hydrogen gas, platinum under acidic conditions, rhodium in neutral or basic conditions, aqueous ruthenium in neutral or basic conditions. It may also be effected using Raney nickel, for example W6 and W7 type, under basic conditions, ambient temperature and atmospheric pressure. Catalytic hydrogenation using ruthenium is preferred since the reaction preferentially occurs in aqueous solvent.
The reduction should be sufficiently mild that carboxyl groups are not reduced. Since sodium borohydride is a comparatively mild reducing agent, the carboxylic acid groups on the dextran molecules will not be reduced. Moreover, carboxylic acids are one of the most difficult of all functional groups to hydrogenate and virtually all other susceptible functionalities should be hydrogenated in preference to them. Accordingly, under most conditions, hydrogenation of carboxylated dextran will not result in conversion of the carboxylic acid groups which are required for complexing.
Other suitable methods of reduction will be apparent to one skilled in the art who will know to avoid conditions which may also reduce carboxyl groups to alcohol groups.
The term carboxylated dextran as used herein refers to dextran containing at least one carboxylic acid group. Methods of preparing carboxylated dextran are described in U.S. Pat. Nos. 3,356,696, 4,370,476 and 4,788, 281, the contents of which are hereby incorporated by reference.
A carboxylic acid group may be introduced into the terminal unit of dextran by hydrolysis of a cyanohydrin intermediate. The cyanohydrin intermediate can be obtained by reacting unhydrogenated dextran containing a terminal aldehyde group with an alkali metal cyanide such as NaCN or, preferably, KCN as follows. Although dextran of any molecular weight may be carboxylated, for use in injectable iron dextran complex, the terminally carboxylated dextran preferably has an average molecular weight (MW) of less than 70,000 and most preferably less than 5,000. 
Dextran may also be oxidized with sodium chlorite in aqueous solution at a pH between 2.5 and 4.5 and at a temperature of about 20xc2x0 to 30xc2x0 C. to form dextran with terminal D-gluconic acid residues having the structure: 
Particularly suited for this method is a low molecular weight dextran of average MW between about 2,000 to 6,000.
Polycarboxylated dextran may be formed as described in U.S. Pat. No. 4,370,476 by first introducing a plurality of aldehyde groups into the internal glucose units of dextran by specific mild oxidation using potassium periodate and then converting the aldehyde groups to carboxylic acid groups by further oxidation or by cyanidation and hydrolysis as follows. If the carboxylated dextran is to be used to complex iron, the average molecular weight of the dextran should be between about 1,000 and 9,000, and preferably between about 1,200 to 6,000 to produce suitable injectable products. 
After reduction of the carboxylated dextran according to the present invention, carboxylated and reduced dextran may be complexed with ferric hydroxide in the known manner to form iron dextran complex. Complex formation is believed to involve carboxyl functionalities and therefore polycarboxylated dextran is believed to be ideally suited for preparing iron dextran complex with a high iron content. Carboxylated and reduced dextran may also be employed to complex with suitable compounds of other metals such as copper or zinc hydroxide where the presence of carboxyl groups facilitates the complexation.
Use of hydrogenated dextran can lessen side reactions associated with injection of dextran. As described in U.S. Pat. No. 2,807,610 to Zief and in J. American Chemical Society, 74, pages 2126-2127 (1952); Zief, M. and Stevens, J. R., hydrogenation is believed to convert, for example, ketone groups and aldehyde groups (including aldehyde interminal positions) and those resulting from rearrangement of cyclic intermediates formed during the hydrolysis of the dextran to the corresponding alcohol group and thereby lessen the possibility of reaction with carbohydrates, proteins or other materials in the blood that may give rise to clinical side effects.
It is believed that during the various synthetic steps required to make carboxylated dextran, some degradation of dextran will occur. The degradation products are likely monosaccharides or other smaller oligomers each of which may contain terminal aldehyde groups. These products may undergo further rearrangement to form hydroxymethylfurfural (HMF) derivatives and other products containing ketone or aldehyde groups. HMF derivatives are very reactive and tend to self polymerise leading to the formation of coloured compounds. Moreover, in the case of polycarboxylated dextran prepared by first introducing a plurality of aldehyde groups into the dextran molecule, it is possible that conversion from aldehyde to carboxylic acid is not complete. For example, some aldehyde groups may be protected by steric hindrance caused by branched side chains, and aldehyde groups may remain in the polycarboxylated dextran. The presence of aldehyde groups is known to be associated with anaphylactic shock.
The chemistry of dextran is complex and reaction side products which may be formed during carboxylation of dextran have not been fully elucidated. However, as shown by a spectrophotometric Reducing Substances Test, described herein, carboxylated and reduced dextran according to the present invention is non-reducing. Reduction of carboxylated dextran is believed to provide carboxylated dextran with reduced side effects on administration and improved physical and chemical stability by eliminating reactive functionalities, examples of which are as follows: 